1. Field of the Invention
The present invention relates to an improved method of isolating a purified α-N-acetylgalactosaminidase from Clostridium perfringens to be used in the conversion of erythrocytes to type O cells to render the cells useful for transfusion therapy.
2. Description of Related Art
The A, B, and H antigens are a clinically significant blood group (Landsteiner, 1901; Mollison et al, 1987). These antigens are terminal immunodominant monosaccharides on erythrocyte membrane glycoconjugates (Harmening, 1989). High densities of these epitopes are present on erythrocyte membranes and antibodies bound to these antigens readily fix complement (Economidou et al., 1967; Romano and Mollison, 1987). Because these epitopes are ubiquitous in nature, immuno-potent and naturally occurring, complement fixing antibodies occur in individuals lacking these antigens, and transfusion of incompatible blood results in fatal hemolytic transfusion reactions (Fong et al., 1974; Schmidt, 1980).
Complex sugar chains in glycolipids and glycoproteins have often been implicated in the growth and development of eukaryotes (Wantanabe et al., 1976). In particular, complex sugar chains play an important part in the recognition of self in the immune system (Mollison, 1987). Glycosidases (both exoglycosidases and endoglycosidases) are enzymes which can modify carbohydrate membrane epitopes, thereby modulating the immune response (Goldstein et al., 1982).
U.S. Pat. No. 4,330,619, issued May 18, 1982; U.S. Pat. No. 4,427,777, issued Jan. 24, 1984; and U.S. Pat. No. 4,609,627, issued Sep. 2, 1986, all to Goldstein, relate to the enzymatic conversion of certain erythrocytes to type O erythrocytes. Since type O erythrocytes can be safely transfused into type A, type B, type A,B recipients, as well as O recipients, type O erythrocytes have significant value in transfusion therapy. The above-mentioned U.S. Pat. No. 4,609,627 discloses the conversion of certain sub-type A and A, B erythrocytes to type O erythrocytes utilizing an α-N-acetylgalactosaminidase fraction from fresh chicken livers. The patent also discusses the significant potential of such enzymes to be used in the conversion of type A2 erythrocytes to type O erythrocytes or type A2 erythrocytes to type B erythrocytes.
The (α-N-acetylgalactosaminidase from domestic chickens is such an enzyme as described above which degrades the human blood group A epitope (Hata et al., 1992). Degradation of the blood group A antigen produces the H antigen, also known as blood group O. Blood group O red blood cells are generally universally transfusable within the ABO blood group system.
The enzyme α-N-acetylgalactosaminidase [EC 3.2.1.49] is a class of exoglycosidases that have been purified from both procaryotes and eucaryotes (McGuire et al., 1972; McDonald et al., 1972; Kadowaki et al., 1989; Itoh and Uda, 1984; Nakagawa et al., 1987; Kubo, 1989; Weissman et al., 1969; Weissman, 1972). Despite the use of this enzyme because of its ready availability from the livers of domesticated chickens, the use is limited. This is based on the lack of published reports regarding the use of commercially useful purification methods for making preparations having no detectable protease or other glycosidase activities along with proven homogeneity (Goldstein, 1984). The low pH optimum of chicken liver enzyme makes it less useful because large masses of enzyme are required. Additionally, red cells must be extensively washed to lower the pH so that the enzyme can efficiently convert red cells from type A to type O or type A2B to type B. Similar problems exist with preparations from other sources including Clostridium perfringens (McGuire et al, 1972). Critically, without a commercially viable method to provide enzymatic activity free of extraneous proteases, neuraminidsae, and glycosidases, there is limited commercial value since the use of a nonhomogeneous enzyme preparation has the potential to damage erythrocyte membranes, therefore leading to poor in vivo viability. It would be particularly advantageous to be able to a isolate α-N-acetylgalactosaminidase from Clostridium perfringens free of contaminants, particularly neuraminidase, while also having additional properties.
It would therefore be useful to develop an α-N-acetylgalactosaminidase which is homogeneous and capable of enzymatic activity against the blood group A epitope.